The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro.
The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro.
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DOI：
10.1093/nar/gkt060
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发表时间：
2013-04-01
影响因子：
14.9
通讯作者：
Dziembowski A
中科院分区：
文献类型：
--
作者：
Drazkowska K;Tomecki R;Stodus K;Kowalska K;Czarnocki-Cieciura M;Dziembowski A
RNA外泌体是参与RNA加工和降解的必需核糖核酸酶复合物。它由一个9-亚基催化惰性环组成，该环由形成中央通道的六个RNase PH样蛋白和三个帽亚基组成，其中KH/S1结构域位于顶部。酵母外泌体催化活性由Dis 3（也称为Rrp 44）蛋白和核特异性核酸外切酶Rrp 6提供，Dis 3蛋白具有内切和外切核糖核酸水解活性。体外研究表明，底物通过环通道后到达Dis 3外切核酸水解活性位点，但缺乏体内支持。在这里，我们构建了一个Rrp 41环亚基突变体与部分封闭的通道，导致热敏性和合成的致命性与Rrp 6缺失。Rrp 41突变引起细胞核和细胞质外泌体底物的积累，包括不停止衰变报告基因，其降解依赖于内切或外切Dis 3活性。这表明中央通道也控制核酸内切活性。使用从重组亚基重构的嗜热毛壳菌外泌体进行的体外实验证实了这一观点。最后，我们分析了Rrp 4帽亚基中保守碱性残基的致死突变的影响，发现它抑制单链和结构化RNA底物的消化。
The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.
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影响因子：
16.8
作者：
Schaeffer, Daneen;Tsanova, Borislava;Barbas, Ana;Reis, Filipa Pereira;Dastidar, Eeshita Ghosh;Sanchez-Rotunno, Maya;Arraiano, Cecilia Maria;van Hoof, Ambro
通讯作者：
van Hoof, Ambro
影响因子：
3.3
作者：
Parker, Roy
通讯作者：
Parker, Roy
影响因子：
11.4
作者：
Tomecki, Rafal;Kristiansen, Maiken S.;Jensen, Torben Heick
通讯作者：
Jensen, Torben Heick
影响因子：
64.5
作者：
Mnaimneh, S;Davierwala, AP;Hughes, TR
通讯作者：
Hughes, TR
影响因子：
64.8
作者：
Frazao, Carlos;McVey, Colin E.;Carrondo, Maria A.
通讯作者：
Carrondo, Maria A.